T cell-mediated immune responses require contact-dependent information exchange between T cells and antigen (Ag)-presenting cells (APC). Naive T cells are primed by mature dendritic cells (DC) in secondary lymphoid organs, such as peripheral lymph nodes (PLN). After a few days of activation, the proliferating T cells differentiate into cytotoxic effector cells (CTL). which can kill APC. CTL activity is thought to be controlled by several mechanisms, including the action of regulatory T cells (Treg) and the propensity of CTL to undergo apoptosis upon withdrawal of survival signals. Thus, after the height of a CTL response, the pool of Ag-specific T cells contracts, leaving behind a small population of long-lived memory cells, which respond more vigorously than naive T cells when the Ag returns. It is widely held that the career decisions taken by T cells are regulated by the spatio-temporal arrangement of interacting communication molecules on the surface of T cells and APC. However, the physical nature and the kinetics of T cell-APC interactions in PLN are still largely unexplored. In preliminary work for this project, we have developed a new multiphoton intravital microscopy (MP-IVM) model to study APC and TCR transgenic CDS T cells in intact popliteal LN of anesthetized mice. This imaging approach produces 3D time-lapse movies of interacting cells at subcellular resolution and will be used to address the following two specific aims: 1.) To analyze the spatial, temporal and behavioral relationship between CTL and APC in PLN and 2.) To explore the effect of Ag-specific Treg on CTL differentiation and function. The proposed experiments will generate a comprehensive, mechanism oriented analysis of CTL differentiation, function and regulation. This information may lead to improved strategies for clinical immunomodulation, e.g. for vaccinations, tumor therapy and treatment of infectious, inflammatory and autoimmune diseases.